HORSERADISH-PEROXIDASE PHE(172)-]TYR MUTANT - SEQUENTIAL FORMATION OFCOMPOUND-I WITH A PORPHYRIN RADICAL-CATION AND A PROTEIN RADICAL

A gene coding for the F172Y mutant of horseradish peroxidase isozyme C (HRP) has been constructed and expressed in both Spodoptera frugiperd a (SF-9) and Trichoplusia ni egg cell homogenate (HighFive(R)) cells. Homology modeling with respect to three peroxidases for which crystal structures are available places Phe(172) on the proximal side of the h eme in the vicinity of porphyrin pyrrole ring C. The pH optimum and sp ectroscopic properties of the F172Y mutant are essentially identical t o those of wild type HRP. V-max values show that the mutant protein re tains most of the guaiacol oxidizing activity. Stopped flow studies in dicate that Compound I is formed with H2O2 at the same rate (k(1) = 1. 6 x 10(7) M(-1) s(-1)) at both pH 6.0 and 8.0 as it is with the wild t ype enzyme. This Compound I species decays rapidly at a rate k(2) = 1. 01 s(-1), pH 7.0, to a second two-electron oxidized species that retai ns the ferryl (Fe-IV = O) absorption. EPR studies establish that a fer ryl porphyrin radical cation is present in the initial Compound I, but electron transfer from the protein results in formation of a second C ompound I species with an unpaired electron on the protein (presumably on Tyr(172)). The presence or absence of oxidizable amino acids adjac ent to the heme is thus a key determinant of whether the second oxidat ion equivalent in Compound I is found as a porphyrin or protein radica l cation.